Project Summary: Knowing the structures of proteins is a linchpin of modern biomedical research, and protein crystallography is the source of over 90% of known structures. A recognized limitation of crystallography is that many important proteins ? including 95% of integral membrane proteins ? do not crystallize, so their structures cannot be determined by crystallography. We propose here to continue our approach to bypass the crystallization hurdle in structure determination of biological macromolecules. Our approach is named serial single molecule electron diffraction imaging (ss-EDI). The envisioned apparatus will take nanomoles of protein solutions and obtain in a few hours data that can enable structure determination at resolutions approaching 1 . The procedure of ss-EDI starts with electrospray ionization (ESI) of proteins from an appropriate solution. The chosen ions with the desired mass-to-charge ratio (or even conformation) are then embedded in superfluid helium droplets for cooling prior to orientation by an electric field and alignment by a laser field ? the fields essentially constitute a ?molecular goniometer? for diffraction. Electron diffraction patterns from individual protein doped droplets all oriented in the same direction are accumulated as the experiment repeats 10 ? 20 times per second. High quality diffraction patterns from different orientations are obtained under different polarization settings of the alignment laser. Using the oversampling method for iterative phase retrieval, electron density maps of the diffraction molecules can then be generated for structure determination. The previous grant period has seen superb progress along two largely independent halves of the project. We have now experimental evidence that electron diffraction of molecules embedded in superfluid helium droplets is a viable path for obtaining atomic structures with sub-Angstrom resolution. Moreover, doping protein ions from an electrospray ionization source into superfluid helium droplets can achieve ion counts up to ~10,000/pulse. Building on these two major advances, key next steps will be to develop the diffraction half of the instrument by demonstrating electron diffraction of gold nanoclusters embedded in superfluid helium droplets and aligned by a laser field. This effort will be sufficient as a proof- of-concept success of ss-EDI, albeit with a highly diffracting sample. In addition, we will also address the question of how protein conformation is influenced by ESI volatilization by monitoring the fluorescence from green fluorescent protein as it traverses the ion delivery path. In the final stage of this work, we will combine the two halves into a complete ss-EDI instrument and apply it to several model proteins including superoxide dismutase, carbonic anhydrase and calmodulin. Alongside the instrument development, we will further develop the image processing and structure refinement software. The impact of this instrument on biomedical research will be tremendous. The instrument will reshape the landscape of structural biology, transform structure-based drug screening, and rapidly determine effects of mutations and deletions on structure. It will also offer rapid structural assessment of individual components in polydispersed mixtures of nanomaterials important for biomedical applications. Furthermore, the ?molecular goniometer? central to ss-EDI as well as other innovations will provide new tools that will find applications in other types of diffraction and imaging methods.